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Hindawi Publishing CorporationJournal of Marine BiologyVolume 2012, Article ID 370247, 13 pagesdoi:10.1155/2012/370247Research ArticleGametogenesis and Spawning of Solenastrea bournoni andStephanocoenia intersepta in Southeast Florida, USAJenna R. Lueg,1 Alison L. Moulding,1 Vladimir N. Kosmynin,2 and David S. Gilliam11 Oceanographic Center, Nova Southeastern University, 8000 N. Ocean Drive, Dania Beach, FL 33004, USA2 Bureau of Beaches and Coastal Systems, Florida Department of Environmental Protection, 3900 Commonwealth Boulevard, MS 49,Tallahassee, FL 32399, USACorrespondence should be addressed to David S. Gilliam, gilliam@nova.eduReceived 24 April 2012; Accepted 26 June 2012Academic Editor: Baruch RinkevichCopyright © 2012 Jenna R. Lueg et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.This study constitutes the first report of the gametogenic cycle of the scleractinian corals Solenastrea bournoni and Stephanocoeniaintersepta. Tissue samples were collected near Ft. Lauderdale, Florida, USA between July 2008 and November 2009 and processedfor histological examination in an effort to determine reproductive mode and potential spawning times. Both S. bournoni and S.intersepta are gonochoric, broadcast spawning species. Gametogenesis of S. bournoni began in April or May while S. intersepta hada much longer oogenic cycle that began in December with spermatogenesis beginning in July. Though spawning was not observedin situ, spawning was inferred from the decrease of late stage gametes in histological samples. In addition, histological observationsof oocyte resorption and released spermatozoa were used to corroborate spawning times. Data indicate that S. bournoni spawns inSeptember while S. intersepta spawns after the full moon in late August or early September.1. IntroductionReproduction of scleractinian corals is one of the mostimportant processes influencing their continued existence[1]. Sexual reproduction is the first step in establishingnew colonies necessary for repopulating degraded reefs. Itcreates genetic variability essential for adapting to changingenvironmental conditions. Information on the reproductivebiology and ecology of corals is critical for understandingtheir distribution, evolutionary mechanisms [2, 3], and formanagement and restoration of damaged areas [4].For broadcast spawning species, synchronous maturationand release of gametes is essential for successful fertilization[5]. Coral gametogenesis and spawning are thought to bedriven by a number of environmental cues including seatemperature, day length, moonlight tidal cycles, and daylightcycles [6, 7], but the exact association remains unknown[1, 7, 8]. For a number of species, temperature defines theseason and month of spawning and timing tends to correlatestrongly with lunar phase [5]. Sunset time can define thehour of spawning [6, 9, 10] and tidal fluctuations maydecrease water movement and lead to increased fertilizationopportunities [11]. It is very likely that coral gametogenesisand spawning are not prompted by a single environmentalcue, but rely on more than one environmental signal [12].In the last few decades, there have been numerous studieson scleractinian coral reproduction [4, 7, 13–16]. About 60%of the approximate 60 known Caribbean species have beeninvestigated [3]. Species of particular interest in southeastFlorida include Solenastrea bournoni and Stephanocoeniaintersepta because of their local abundance. Stephanocoeniaintersepta and Solenastrea bournoni are common speciesfound offshore in all three counties (Miami-Dade, Broward,and Palm Beach) and on all reef types (nearshore ridgecomplex, Inner reef, Middle reef, and Outer reef) within thesoutheast Florida region [17, 18]. These species are not ascommon in the rest of the Caribbean where S. interseptagenerally comprises less than 5% of the coral populationin areas such as the US Virgin Islands, Venezuela, Turksand Caicos, Mexico, Belize, and Bahamas and S. bournoniis even less abundant [19]. There is no information in thepublished literature on reproductive mode or spawning timeof S. bournoni. Stephanocoenia intersepta has been reportedto be a gonochoric species that has been observed broadcast2 Journal of Marine BiologyTable 1: Criteria for differentiating oocyte and spermary developmental stage based on Szmant-Froelich et al. [27].Stage Oocytes SpermariesI Nucleus large, located in or adjacent to mesoglea Small cluster of ≤10 cells surrounded by mesogleaII Accumulation of cytoplasm around nucleus, located in mesoglea Larger cluster of ≥10 cellsIII Increased amount of cytoplasm, but no vitelline membrane Cells closer together, central lumen developedIV Larger, elongated and presence of vitelline membrane Cells very small, undergoing meiosis, tails in lumenspawning in Bonaire 3–7 nights after the August full moon[16] and in the Gulf of Mexico from 6 to 11 nights afterthe August or September full moon between the hours of19:30 and 20:00 [9, 20]. Females of this species have beenobserved releasing a high percentage of fertilized eggs [20,21] and it has been proposed that eggs are fertilized in thetentacles just prior to release [21]. Cycles of oogenesis andspermatogenesis have not been previously described for thesetwo species. The purpose of this study was to document thegametogenic cycle of S. bournoni and S. intersepta and todetermine their spawning times in southeast Florida.2. Materials and Methods2.1. Collection and Histological Processing. Solenastreabournoni and S. intersepta were sampled twice per monthfrom July 2008 through July 2009 and weekly in August andSeptember 2009. During each collection date, five coloniesof S. bournoni and S. intersepta were sampled. Additionally,five S. bournoni samples were collected twice per week inOctober and November 2009. Colonies were sampled insoutheast Florida near Ft. Lauderdale (26◦03′N to 26◦19′N)in 4 to 18 m depth. Tissue samples measuring approximately6–12 cm2 in size were removed using a hammer and chisel ora 2.5 cm steel core. All samples were taken from the middle ofthe colony to avoid less fecund edges [22–24]. Only coloniesthat had a diameter of over 10 cm were sampled to ensuresexual maturity [14, 24–26] and each colony was sampledonly once. Epoxy was placed on the exposed skeleton wheretissue was removed to minimize potential settlement of algaeand boring organisms. Collection methods caused minimaldamage to S. bournoni colonies since tissue completelyregrew over the sample area within 6 months. However, S.intersepta had much slower regeneration rates and showedminimal regrowth over the sampled areas during the studyperiod. Collected samples were fixed immediately in 10%aqueous zinc-buffered formalin (Z-fix) for 24 hours. Tissuesamples were decalcified using a buffered 10% hydrochloricacid solution, dehydrated using a series of ethanol andxylene, infiltrated with paraffin, and embedded in paraffinblocks.Serial sections of 5 µm were cut with a microtomeat three depths of the tissue and placed on slides. Thefirst serial sections were taken at a location just belowthe actinopharynx, and subsequent sections were takenapproximately 0.15–0.3 mm and 0.45–0.6 mm below theactinopharynx. Two replicate slides were made at each of thethree depths, yielding 6 slides for each sample. Three of theseslides, one at each depth, were stained with Heidenhain’sStage I020406080100Stage IIMean (±SE) (%)020406080100Stage III2008020406080100Stage IV2009Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 020406080100Solenastreabournoni oocytesFigure 1: Solenastrea bournoni mean (±SE) percentage of oocytesper colony quantified through histological examination.azocarmine aniline blue to highlight reproductive structures.The rest were stained with Harris’ hematoxylin and eosine(H&E) in order to confirm the presence of released sper-matozoa in which the nuclei stain dark purple. All stainingcharacteristics described are with Heidenhain’s azocarmineaniline blue unless otherwise noted.2.2. Quantification of Gametes and Fecundity. Gamete devel-opmental stages were determined from the slide that con-tained the most gametes which was usually the deepest slide.Criteria determining gamete developmental stage (Table 1)were modified from Szmant-Froelich et al. [27]. Using theslide with the most gametes, the percentage per colonyof each gamete stage was calculated per sampling dateusing the total number of gametes in 7–10 polyps percolony. Resorption and released spermatozoa percentagesper sampling date were calculated based on the numberof mesenteries of 7–10 polyps per colony that containedresorbed oocytes or spermatozoa released from spermaries.Polyp fecundity was calculated for every female colonysampled during the month prior to spawning. Fecunditywas calculated by multiplying the mean number of mature(stage IV) oocytes in cross sections of ten polyps per colonyand the mean number of mature oocytes in longitudinalsections of 10 mesenteries, yielding mean stage IV oocytesJournal of Marine Biology 3I(a)III(b)III III(c)IVIIIII(d)IVViSc(e)RIV(f)Figure 2: Photomicrographs of Solenastrea bournoni oocyte stages I–IV (defined by Table 1). (a) Stage I oocyte; (b) stages I and II oocytes;(c) stage III oocytes; (d) stages II, III and IV; (e) stage IV oocytes, Vi = vitelline membrane, Sc = scalloping of nucleus; (f) stage IV oocyteadjacent to oocyte resorption (R). All scale bars 20 µm except for (c) which is 100 µm.per polyp. For description of developmental stages, meanoocyte diameters were measured from multiple colonies overseveral sampling dates when abundance was highest. Meanabundance of stage IV oocytes per colony was calculated andcompared to environmental factors including moon phaseand temperature. Chi-squared tests were used to determinesex ratio significance.2.3. Environmental Data. Temperature loggers weredeployed at 10 meters depth where most coral sampleswere collected. Regression analyses were preformed toexamine the relationship between oocyte abundance andtemperature. Stage IV oocyte abundance was plotted withmoon phase to determine if spawning times could be linkedto lunar cycles.3. Results3.1. Solenastrea bournoni. Solenastrea bournoni is a gono-choric species with a 1 : 1 sex ratio (χ2= 0.157, df = 1,P = 0.692). A total of 112 females and 118 males weresampled, and seven colonies did not have gametes and4 Journal of Marine BiologyStage II020406080100Stage III2009020406080100Stage IV2008Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 020406080100Stage IMean (±SE) (%) 020406080100Solenastrea bournoni spermariesFigure 3: Solenastrea bournoni mean (±SE) percentage spermarystages per colony quantified through histological examination.could not be determined to be male or female. Out of237 colonies sampled, there were two male colonies thatcontained a single mature oocyte in the same mesentery thatcontained spermatocytes and one male colony that containedtwo oocytes in separate polyps surrounded by maturespermatocytes. No larvae were seen in any colony, and latestage oocytes did not contain zooxanthellae. Fecundity wascalculated as 166.34 ± 42.00 (SE) oocytes per polyp (n =8 colonies) in 2008 and 305.33 ± 78.48 oocytes per polyp(n = 10) in 2009.3.1.1. Gametogenesis. Oogenesis in Solenastrea bournonibegan in April, and stage I oocyte abundance peakedfrom April to early May (Figure 1). Mean diameter ofstage I oocytes observed during their peak abundance was25.24 ± 0.98µm and ranged in size from 16 to 30 µm(n = 26 oocytes). Early stage I oocytes were located in thegastrodermal layer and migrated into the mesoglea (Figures2(a) and 2(b)). They were characterized by large nuclei inrelation to the amount of cytoplasm. Nuclei stained lightpink to grey with a magenta nucleolus, and the cytoplasmstained light grey.Stage II oocytes were first observed in early June(Figure 1) but may have developed earlier (no females werecollected in late May). Highest percentages per colony wereseen in early June. Mean diameter of randomly chosen stageII oocytes from June samples was 57.32 ± 2.83µm andranged from 35 to 98 µm (n = 30). These larger oocytes werelocated in the mesoglea and stained light grey with a lightpink nucleus and magenta nucleolus (Figure 2(b)). The sizeof cytoplasm was greater than the nucleus.Stage III oocytes began to appear in mid-June, andabundance peaked in mid-July 2008 and in early August 2009(Figure 1). Stage III oocytes measured during this periodwere larger than stage II with a mean diameter of 102.12 ±3.65µm and a range of 69–149 µm (n = 30). Stage III oocytesstained a darker pink in color and had a centrally locatednucleus (Figures 2(c) and 2(d)).Stage IV oocytes developed by mid-June, peaked inAugust, and continued to comprise a high percentage oftotal oocytes through the end of sampling in late November(Figure 1). Mean diameter of stage IV oocytes was 225.59 ±10.18µm and ranged from 153 to 341 µm (n = 30). StageIV oocytes were characterized by their large size and darkvitelline membrane (Figure 2(d)). Depending on the section,the nucleus had usually migrated to one side, and thevitelline membrane sometimes had a scalloped appearance(Figure 2(e)). Stage IV oocytes also had an increased amountof lipid vacuoles that stained orange, pink, magenta, andlight purple.Resorption of oocytes was observed throughout mostof the year in 2009 although in small percentages. Inearly resorption (Figure 2(f)), the vitelline membrane brokeapart, and lipid vacuoles were clumped together. Lipid vac-uoles slowly dispersed into the gastrodermis and degraded.Resorption after February was seen in less than 1% of allmesenteries examined. In July, there was a low occurrenceof resorption (4.46% of mesenteries in all colonies sampledthat month), and in early October one colony showedresorption of oocytes in 20% of its mesenteries. Higher levelsof resorption were observed through the end of sampling inlate November.Spermatogenesis began rapidly in May with all stagesappearing in the same two-week period (Figure 3). StageI and stage II spermaries stained light pink and weredifferentiated by the number of cells (Figures 4(a) and 4(d)).Stage I spermaries were characterized by fewer than 10 small,loosely clustered cells located in the mesoglea, and stageII spermaries contained more than 10 cells. Both stage Iand stage II spermaries had the highest percentages in May(Figure 3).Stage III spermaries peaked in mid-July (Figure 3)and were comprised of larger and darker cells that werelocated closer together around a centrally located lumen(Figures 4(b) and 4(d)). The highest percentages of stage IVspermaries were in October, and they were observed throughFebruary (Figure 3). The magenta cells were very small, andspermatozoa tails were lined up in the lumen (Figures 4(c)and 4(d)).Low percentages of released spermatozoa were observedin the gastrodermis during many of the sampling dates(Figures 4(d) and 4(e)). Small spikes in percentage of releasedspermatozoa occurred in mid-August and October 2008followed by the highest percentage observed in November. In2009, released spermatozoa were seen in variable percentagesbetween colonies starting in January. Percentage of releasedspermatozoa decreased greatly in May, but they were stillobserved in small percentages until July. Then percentagesincreased at the end of September, continuing throughNovember to near 100%. In stains of hematoxylin andeosine (H&E), the heads of the spermatozoa stained darkpurple shortly after being released (Figure 4). Months later,Journal of Marine Biology 5III(a)IIIIII(b)IVSTIV(c)IIIIIIVR(d)R(e)R(f)Figure 4: Photomicrographs of Solenastrea bournoni spermary stages I–IV (defined by Table 1). (a) Stages I and II spermaries; (b) stageIII spermaries; (c) stage IV spermaries, ST = spermatozoa tails lined up; (d) stages II, III, and IV, R = spermatozoa being released intogastrovascular cavity; (e) released spermatozoa (R) stained with Heidenhain’s azocarmine aniline blue; (f) released spermatozoa (R) stainedwith H&E. All scale bars 20 µm except for (c) which is 10 µm.residual spermatozoa did not stain purple, indicating theyhad degraded and were no longer viable.3.1.2. Lunar Periodicity. Mean abundance of stage IV oocytesper colony was plotted with lunar phase (Figure 5). Peakabundances of stage IV oocytes occurred in late Augustand early September and experienced the largest decreasebetween the full and new moon of September. Smallerdecreases in abundance were also observed between the fulland new moons of October though few female colonies weresampled before the October 2008 full moon and after theOctober 2009 full moon.3.1.3. Temperature. Mean stage IV oocyte abundance trackedmean daily water temperatures (Figure 6) with oocyte abun-dance decreasing and increasing in unison with temperature.In 2009, peaks in oocyte abundance in July, September,and August occurred after peaks in temperature. To testthis correlation, a simple linear regression was performedplotting all stages of oocyte abundance against temperature(Figure 7). A significant relationship was found betweenoocyte abundance and temperature (R2= 0.67, P < 0.001),so the same analysis was performed for all stages of spermaryabundance (Figure 8). A significant relationship was alsofound between spermary abundance and temperature (R2=0.65, P < 0.001).6 Journal of Marine Biology2008Aug Sep Oct Nov Dec0100200300400Mean (±SE) stage IVoocyte abundance colony−1(a)Oocyte abundanceMoon phase2009Aug Sep Oct Nov Dec0100200300400500Mean (±SE) stage IVoocyte abundance colony−1(b)Figure 5: Solenastrea bournoni mean (±SE) abundance of stage IVoocytes per colony at each sampling date with moon phases foryears 2008 and 2009; full moons represented by peaks in moonphase line.Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 01002003004005006001820222426283032Oocyte abundanceMean daily temperatureMean (±SE) stage IVoocyte abundance colony−1Temperature (◦C)Figure 6: Solenastrea bournoni mean (±SE) abundance of stageIV oocytes per colony at each sampling date with temperature forsampling dates 2008 through 2009.3.1.4. Predicted Spawning Times. Results indicate that Sole-nastrea bournoni spawned after the full moon of September.In 2008, the greatest decrease in mean oocyte abundancewas observed between 6 and 29 September, from 338 to 173oocytes per colony. The full moon occurred on September15. Oocyte resorption was first observed in October inOocytes20 22 24 26 28 30 320100200300400500Temperature (◦C)y = −1017.36 + 46.23xR2= 0.67Oocyte abundance colony−1Figure 7: Simple regression of temperature and Solenastreabournoni oocyte abundance, including all stages per colony at eachsample date. R2= 0.67, P < 0.001.Spermaries20 22 24 26 28 30 320200400600800100012001400y = −2748.03 + 124.09xR2= 0.65Spermary abundance colony−1Temperature (◦C)Figure 8: Simple regression of temperature and Solenastreabournoni spermary abundance, including all stages per colony ateach sample date. R2= 0.65, P < 0.001.low percentages, and percentages increased in November.Released spermatozoa were observed in August but were notpresent again until October. A greater amount of oocyteresorption and released spermatozoa in October indicatesthat the main spawning event took place before October.In 2009, the highest peak in mean abundance of stage IVoocytes was on 9 September with 505 oocytes per colony. Afull moon occurred on September 4, and mean abundanceof stage IV oocytes decreased after 9 September. Oocyteresorption also started in October 2009. The amount ofreleased spermatozoa was moderate on 17 September, butincreased through December of 2009. Unfortunately, the factthat only one of the five colonies sampled after the potentialspawning time in 2008 was female and only one of the fivecolonies sampled before the potential spawning in 2009 wasfemale precludes statistical analysis of significant decreases inoocyte abundance to support the proposed spawning period.However a similar decrease in mature oocyte abundanceafter the full moon of September in both years, coupledJournal of Marine Biology 7Stage III2008020406080100020406080100Stage IV2009Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct 020406080100Stage IStage IIMean (±SE) (%)020406080100Stephanocoenia intersepta oocytesFigure 9: Stephanocoenia intersepta mean (±SE) percentages ofoocyte per colony quantified through histological examination.with observed oocyte resorption and increases of releasedspermatozoa in October, indicates the main spawning eventlikely occurred after the full moon of September.3.2. Stephanocoenia intersepta. No cases of hermaphroditismwere observed in 181 colonies sampled over 15 months.Sixty-five colonies were found to contain no gametes. Nosex could be assigned to 31 of these 65 colonies because11 had no gametes during the reproduction period and 20were sampled during the winter nonreproduction period.The remaining 34, sampled from January to June, wereassumed to be males that had not yet developed gametessince spermatogenesis was not observed until July. Sex ratiowas 1 : 1 (78 females, 72 males, χ2= 0.24, df = 1, P > 0.01).Female fecundity of S. intersepta was 64.51 ± 12.04 oocytesper polyp in 2008 (n = 8) and 92.67±9.27 oocytes per polypin 2009 (n = 19).3.2.1. Gametogenesis. Oocytes were observed in samplesof S. intersepta from December through October whilespermatocytes were present from late July through October.Resorption of oocytes was present for 2 to 3 months inSeptember to early November. Released spermatozoa werepresent in September and October, but by mid-November,no gametes or remnant gametes were seen.Stage I oocytes were first observed in mid-December ina longitudinal section of one colony but were not seen inany cross sections which were used to quantify gamete stages.Stage I oocytes peaked in percentage in January and wereobserved through August 2009 in low percentages (Figure 9).Stage I oocytes randomly chosen during January throughAugust had a mean diameter of 15.81 ± 0.84µm and rangedfrom 9.9 to 28.86 µm (n = 30 oocytes). These oocytes werefound in the gastrodermis adjacent to mesoglea or in themesoglea (Figure 10(a)). They had large nuclei in relation tothe amount of cytoplasm, and the nuclei stained pink with amagenta nucleolus. If cytoplasm was seen, it was light pinkor grey in color.Stage I oocytes developed into stage II oocytes as earlyas December but were seen only in a longitudinal sectionof onecolony. The highest percentage of Stage II oocyteswas observed in April (Figure 9). Stage II oocytes randomlychosen from all dates observed had a mean diameter of47.81 ± 2.09µm, and they ranged in size from 31.33 to63.71 µm (n = 30). Nuclei stained grey or light pink, andcytoplasm stained grey. Stage II oocytes were larger withthe amount of cytoplasm greater than the nucleus, and theyresided in the mesoglea (Figure 10(b)).Stage III oocytes developed in April, and highest percent-ages were present in late June (Figure 9). The mean diameterof Stage III oocytes was 140.62 ± 5.69µm and rangedfrom 79.86 to 206.3 µm (n = 32). Cytoplasm stained lightpink, and oocytes usually had a centrally located nucleus(Figure 10(c)).Stage IV oocytes first developed in mid-July, peaked inpercentage in August, and declined after September (Fig-ure 9). Stage IV oocytes randomly chosen from July throughSeptember had a mean diameter of 318.84 ± 13.78µm andranged from 184 to 435.2 µm (n = 30). The lipid vacuolesin the cytoplasm were more defined than they were in stageIII and stained pink (Figure 10(d)). The vitelline membranestained blue to purple and appeared scalloped in late stage IVoocytes (Figure 10(d)).In 2008, resorption of S. intersepta oocytes occurredfrom early September through late October with no signsof resorption in November. In 2009, oocyte resorption wasseen in low percentages in late July through early September,but in mid-September, oocyte resorption was at 100% in allmesenteries.Stage I spermaries formed in late July, and the highestpercentage was seen in August (Figure 11). Stage I spermarieswere characterized by a small cluster of less than 10 looselypacked cells that stained light pink and were found in themesoglea (Figure 12(a)). Stage II spermaries were observedin late July to early August with the highest percentagesoccurring in September 2008 and late August 2009 (Fig-ure 11). Stage II spermaries stained light pink and consistedof more than 10 loosely packed cells (Figure 12(b)). Stage IIIspermaries were first observed in late July to early August,and peak percentages occurred in August (Figure 11). StageIII spermaries were larger with more compact, darker stain-ing cells and had a centrally located lumen (Figure 12(c)).Stage IV spermaries were first observed in August, andthe highest percentages occurred in early August 2008 andearly September 2009 (Figure 11). Spermatocytes of stageIV spermaries stained dark magenta with pink staining tails(Figure 12(d)).Released spermatozoa appeared in late September 2008and were at their highest percentage in late October (Fig-ure 11). In 2009, spermatozoa were observed outside of themesoglea in early September, and in mid-September (Figures12(e) and 12(f)) they were observed in almost at 60% of allmesenteries counted.8 Journal of Marine BiologyI(a)II(b)III(c)IVIV(d)IVViSc(e)RN(f)Figure 10: Photomicrographs of Stephanocoenia intersepta oocyte stages I–IV (defined by Table 1). (a) Stage I oocytes; (b) stage II oocyte;(c) stage III oocyte; (d) stage IV oocytes; (e) stage IV oocyte, Vi = vitelline membrane, Sc = scalloping of vitilline membrane; (f) oocyteresorption (R), N = Nucleus. All scale bars 20 µm except (d) which is 100 µm.3.2.2. Lunar Periodicity. Stephanocoenia intersepta meanabundance of stage IV oocytes per colony on each samplingdate was plotted with lunar phase (Figure 13). In 2008,there was a decrease in abundance between 26 August and 6September which occurred after the full moon of 16 August.A decrease in stage IV oocytes occurred after 9 September2009 following the full moon on 4 September.3.2.3. Temperature. Oogenesis began during a time ofcooler water temperatures in December. Abundance of allspermary stages was plotted against temperature, and alinear regression analysis indicated a very low correlationthat was not significant (R2= 0.09, P = 0.17). Howevermaximum abundance of late stage gametes occurred duringthe warmest time of the year.3.2.4. Predicted Spawning Times. In 2008, highest abundanceof stage IV oocytes was 10 days after the full moon of 16August. Results of a Mann-Whitney rank-sum test indicateda significant difference (P < 0.013) in abundance of stageJournal of Marine Biology 9Mean (±SE) (%) 020406080100 Stage I020406080100Stage III2008020406080100Stage IV2009Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct 020406080100Stage IIStephanocoenia intersepta spermariesFigure 11: Stephanocoenia intersepta mean (±SE) percentages ofspermaries per colony quantified through histological examination.IV oocytes between colonies collected on 26 August and6 September 2008, supporting the idea that S. interseptaspawned at least 10 days after the August 2008 full moon.In 2009, the greatest decrease in abundance of stage IVoocytes occurred between 9 September and 17 September.Unfortunately, only one female colony was sampled on 17September which was the last sampling date. This colonyshowed oocyte resorption in 100% of all mesenteries exam-ined. The four male colonies sampled on this date releasedspermatozoa in a mean of 60% of examined mesenteries,indicating a spawning event likely occurred before this date.These data indicate that S. intersepta spawned between 9 and17 September 2009, following the full moon of 4 September.4. DiscussionGametogenic cycles of S. bournoni and S. intersepta differedgreatly even though they likely spawned during similartimes of the year. Most Caribbean broadcast spawning coralspecies studied to date begin gametogenesis a few monthsafter spawning [24]. Stephanocoenia intersepta followed thispattern while S. bournoni did not. Gametogenesis of S.bournoni is similar to the Caribbean gonochoric broadcastspawning species Oculina varicosa which starts gametogene-sis in early summer (S. bournoni starts in spring) and spawnsin late summer and fall [8] as S. bournoni does. No reportsof other Caribbean broadcast spawning species that begingametogenesis in spring were found, indicating that this isan unusual occurrence.Gametogenesis was more rapid in S. bournoni than inS. intersepta. Oogenesis in S. bournoni took less than 3months (from early April to late June) to produce fullydeveloped oocytes, but oocytes continued to develop forthree more months through mid-October. The oogenic cycleof S. intersepta began in December, and oocytes slowlymatured over 9 months. Spermaries developed rapidly forboth species. No spermaries were seen in S. bournoni in earlyMay. However, all stages were observed only 12 days later.Stephanocoenia intersepta spermaries took only one monthto mature after spermatogenesis began in mid-to late July.Temperature has been thought to play a potential role ininducing gametogenesis [28], and many Caribbean broadcastspawning species release their gametes during the highestannual water temperatures [20, 24]. Gametogenesis in S.bournoni was significantly correlated with temperature, andincreases and decreases of S. bournoni oocyte abundancefollowed the temperature regime. These data suggest thatthe trend of warming or the change in temperature maytrigger S. bournoni to begin gametogenesis. Stephanocoeniaintersepta gametogenesis did not show a relationship withincreasing temperature indicating that there may be otherexogenous factors such as moonlight [26] or day length thatcue gamete production. However, similar to other Caribbeancorals, maximum mature gamete abundance coincided withthe warmest time of the year suggesting that temperaturemay play a role in gamete maturation [29].Both species are gonochoric, but three colonies of S.bournoni were found to contain individual cosexual polypsthat functioned predominantly as male but contained onemature oocyteper polyp. This sexual system is calledandromonoecious when male and cosexual polyps are foundon the same colony [30]. When male, female, and cosexualpolyps are found on separate colonies, this sexual system iscalled polygamodioecious, and species that have this patternmay be sequential cosexuals with overlap of alternating cyclesof male and female function leading to cosexual polyps [30].However, because such a small proportion of the coloniessampled (3 out of 237 colonies) exhibited this pattern, a smallnumber of polyps (one or two) in the tissue sample displayedthis pattern, and only one oocyte occurred in each of thefour observed cosexual polyps; it is unlikely that this speciesis sequential cosexual. Thus, S. bournoni can be described asstable gonochoric since it is not unusual to find a low amountof hermaphroditism among gonochoric species [28].Solenastrea bournoni female fecundity was greater thanS. intersepta in terms of the number of oocytes per polyp.The number of stage IV oocytes varied between species likelydue to differences in oocyte and polyp morphology [31].The polyps of S. bournoni were larger than S. intersepta,creating space for a greater number of oocytes. Diameter ofS. intersepta stage IV oocytes was larger than S. bournonipossibly due to a longer period of oogenesis. Fecundity ofS. bournoni varied between years as has been reported inother species [23] and may be a result of the small numberof female colonies sampled prior to spawning. Fecundity ofS. intersepta was not as variable between years.The most widely accepted cue for coral spawning islunar phase [7, 13, 32], and corals have blue-sensitivephotoreceptors that have the ability to detect moonlight [33].Although spawning was not observed directly, histologicalevidence indicated that S. bournoni is a broadcast spawner.No planulae were observed in any of the sampled colonies,and varying stages of gamete development followed by a10 Journal of Marine BiologyI(a)IIIIIIII(b)IIIIII(c)IVIVIVST.(d)R(e)RRM(f)Figure 12: Photomicrographs of Stephanocoenia intersepta spermary stages I–IV (defined by Table 1). (a) Stage I spermary; (b) stage IIspermaries; (c) stage III spermaries; (d) stage IV spermaries, ST = spermatozoa tails lined up; (e) released spermatozoa (R) stained withHeidenhain’s azocarmine aniline blue; (f) released spermatozoa stained with H&E, M = mesoglea. All scale bars 20 µm.sharp decrease in the abundance of mature gametes indicatedspawning activity [24, 34, 35]. Based on results of thisstudy, S. bournoni appears to spawn after the full moon ofSeptember. Although the main spawning event is thoughtto occur in September, it is possible that this species mayalso spawn in August or October due to the presence ofmature gametes during these months and the decreases inabundance of mature oocytes. There was a smaller secondarydecrease in abundance of mature oocytes after the full moonof October, but small samples sizes before and after the fullmoon limited interpretation of the data. It is not unusual fora coral species to spawn over more than one lunar period[14–16, 20, 36] especially if gametogenesis is asynchronous[8]. With its short gametogenic cycle and extended periodswith mature gametes, it may be possible for S. bournoni tospawn over multiple months. Since gametogenesis is closelytied to temperature in this species, spawning may occursooner or over multiple months in areas closer to the equator.However, we do not have data to confirm these suggestions.It is likely that S. intersepta spawned 5–10 days afterthe full moon in August or September depending on howearly the full moon occurred. Histological evidence suggestedthat spawning by S. intersepta occurred at least 10 days afterthe 16 August 2008 full moon and at least 5 days afterthe 4 September 2009 full moon. Spawning could not havebeen successful if eggs were released after the 6 August 2009full moon because there were no mature spermaries untillate August. Reports from Bonaire indicate that S. interseptaJournal of Marine Biology 112008Jul 21 Aug 04 Aug 18 Sep 01 Sep 15050100150200250Mean (±SE) stage IVoocyte abundance colony−1(a)2009Jul 20 Aug 03 Aug 17 Aug 31 Sep 140100200300Oocyte abundanceMoon phaseMean (±SE) stage IVoocyte abundance colony−1(b)Figure 13: Stephanocoenia intersepta mean (±SE) abundance ofstage IV oocytes per colony at each sampling date plotted withmoon phases for years 2008 and 2009; full moons represented bypeaks in moon phase line.spawns between 3 to 7 nights after the August full moon [16].At the Flower Garden Banks in the Gulf of Mexico, a regionwith a difference of only 157 km in latitude from sample sitesin southeast Florida, S. intersepta spawns 7 to 9 days afterthe August or September full moon according to Vize et al.[9] and 6 to 10 days after the August full moon as reportedby Hagman et al. [20]. Thus, the results of the current studyagree with other published records of spawning times fromthe Caribbean and Gulf of Mexico.Resorption of oocytes and released spermatozoa mayhelp to confirm spawning times. Resorption of oocytes isnot fully understood, but it is thought that by breakingdown the large amount of lipid vesicles in oocytes, energycan be absorbed back into the coral [7]. Since oocytesare energetically costly, it would be beneficial to use anyexcess energy from oocytes that were not released duringspawning. Assuming that resorption of oocytes and residualspermatozoa occurs after a spawning event, presence ofresorbed gametes may provide additional evidence of whena spawning event occurred. Resorption in S. bournoni beganin mid-November 2008, indicating that spawning occurredprior to this date. Increased percentages of released sperma-tozoa in October indicate that spawning may have occurredin September. Resorption and release of spermatozoa in 2009occurred through much of the year in S. bournoni. Oocyteswere broken down over a long period of time, and residualspermatozoa that were not viable, as indicated by a lack ofpurple staining with H&E, persisted for a very long time.High amounts of resorption occurred in November 2009,and high percentages of released spermatozoa were observedin mid-September, again supporting the idea that S. bournonispawned in September. In most coral reproduction studies,samples are not taken year round, and no other examplesof this long-term breakdown of gametes were found in theliterature. Oocyte resorption over long periods suggests thatS. bournoni may not immediately require the energy gainedfrom oocyte resorption. Given S. bournoni’s delayed onsetof gametogenesis until spring, perhaps this extra energy isnot needed until production of gametes commences. A slowbreakdown may provide energy over a long period of timeduring winter months when metabolism rates may be slowerwith cooler temperatures.Resorption of S. intersepta oocytes began in August andoccurred in high percentages in September 2008. This obser-vation, coupled with the appearance of a high percentageof released spermatozoa beginning at the end of September,suggests that a spawning event happened before this timeand supports the idea of spawning after the August 2008 fullmoon. All residual oocytes and spermatocytes were gone byearly November. In 2009, oocyte resorption was seen in lowamounts (less than 2%) from July through early September,but on 17 September, the one female colony sampled showedresorption in 100% of mesenteries. Low percentages ofreleased spermatozoa were observed in September until the17th when released spermatozoa were at 60%. This evidencesupports that spawning of S. intersepta occurred between 9and 17 September 2009. After spawning, S. intersepta quicklybroke down leftover gametes. Stephanocoenia intersepta mayneedthis energy to support gametogenesis which began inDecember. The role of resorption in coral energetics and itstiming relative to gametogenesis needs further study.In summary, this study provides the first descriptionof the gametogenic cycles of S. bournoni and S. intersepta.Spawning of S. bournoni is thought to occur in southeastFlorida between 5 to 14 days after the full moon of Septemberwith a possible secondary spawning event occurring after thefull moon of October. Stephanocoenia intersepta is thought tospawn 2 to 12 days after the late August or early Septemberfull moon which is consistent with reports from the Gulf ofMexico and Caribbean. More intense sampling during thesepotential spawning windows is needed to more preciselypinpoint the exact time of spawning, but this study providesa good foundation for further investigation.AcknowledgmentsThis research was supported in part by the Florida Depart-ment of Environmental Protection and the Hillsboro InletDistrict. Coral samples were collected under the FloridaFish and Wildlife Conservation Commission Special ActivityLicense 08RP-1050. Special thanks to Adam T. St. Gelais,and D. Abigail Renegar for their support in laboratorytechniques. Field assistance was provided by Elizabeth A.Larson, Stephanie J. Bush, Vanessa I. P. Brinkhuis, Allison12 Journal of Marine BiologyS. Brownlee, Paola Espitia-Hecht, Daniel P. Fahy, MauricioLopez, Jennifer Mellein, and Zach Ostroff.References[1] J. R. Guest, A. H. Baird, K. E. Clifton, and A. J. 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Thomas,“The reproductive seasonality and gametogenic cycle ofAcropora cervicornis off Broward County, Florida, USA,” CoralReefs, vol. 25, no. 1, pp. 110–122, 2006.Submit your manuscripts athttp://www.hindawi.comHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014 Anatomy Research InternationalPeptidesInternational Journal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporation http://www.hindawi.com International Journal ofVolume 2014ZoologyHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Molecular Biology International GenomicsInternational Journal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014The Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014BioinformaticsAdvances inMarine BiologyJournal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Signal TransductionJournal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014BioMed Research InternationalEvolutionary BiologyInternational Journal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Biochemistry Research InternationalArchaeaHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Genetics Research InternationalHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Advances inVirolog yHindawi Publishing Corporationhttp://www.hindawi.comNucleic AcidsJournal ofVolume 2014Stem CellsInternationalHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Enzyme ResearchHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014International Journal ofMicrobiology
